1. Field of the Invention
The present invention relates to methods of use of facially amphiphilic polyaryl and polyarylalkynyl polymers and oligomers, including pharmaceutical uses of the polymers and oligomers as antimicrobial agents and as antidotes for hemorrhagic complications associated with heparin therapy. The present invention also relates to novel facially amphiphilic polyaryl and polyarylalkynyl polymers and oligomers and their compositions, including pharmaceutical compositions.
2. Background Art
Bacterial drug resistance is a significant current health problem throughout the world. Multiple drug resistance is being commonly seen in a number of human pathogens (Hiramatsu, K., et al., J. Antimicrob. Chemother. 40:311-313 (1998); Montecalvo, M. A., et al., Antimicro. Agents Chemother. 38:1363-1367 (1994); Butler, J. C., et al., J. Infect. Dis. 174:986-993 (1996); Lyytikainen, O., et al., J. Hosp. Infect. 31:41-54 (1995)), and the incidence of drug-resistant hospital infections is growing at a rapid rate. In some U.S. hospitals, nosocomial pathogens, such as E. faecium and Acinetobacter species, have acquired multiple resistance determinants and are virtually untreatable with current antimicrobial agents (Threlfall, E. J., et al., Lancet 347:1053-1054 (1996); Bradley, J. S., and Scheld, W. M., Clin. Infect. Dis. 24 (Suppl. 2):S213-221 (1997)). Bacterial resistance has now reached epidemic proportions and has been attributed to a variety of abuses of antibiotic treatments, including overuse (Monroe, S., and Polk, R., Curr. Opin. Microbiol. 3:496-501 (2000)), inappropriate dosing at sub-therapeutic levels (Guillemot, D., et al., JAMA 279:365-370 (1998)), and misuse as antimicrobial growth promoters in animal food (Lathers, C. M., J. Clin. Pharmacol. 42:587-600 (2002)). The threat of bio-terrorism has provided a further impetus to develop novel classes of antibiotics, particularly ones against which it will be difficult to develop resistant bacterial strains. The pharmaceutical scientific community is responding to this challenge by focusing on the development of new antibiotic drugs. Much of this work, however, is directed to synthesizing analogs of known drugs, such as cephalosporins and quinolones, that, while potentially useful for a short time, will inevitably also encounter bacterial drug resistance and become ineffective. Antibacterial drugs currently represent approximately 65% of the market in infectious disease drugs (Global Information, The world market for anti-infective series: Volume II: The world market for antibacterial medications, Kalorama Information (2003)). Thus, therapeutically effective antimicrobial drugs that act by novel mechanisms would provide an economc as well as a human health benefit.
Following the initial discovery of cecropins and magainins, antimicrobial peptides have become a large and growing class of biologically interesting compounds (Zasloff, M., Curr. Opin. Immunol. 4:3-7 (1992); Zasloff, M., Trends Pharmacol. Sci. 21:236-238 (2000)). These compounds represent the first line of defense against microbes for many species, including plants, insects, worms, and mammals (Boman, H. G., Immunol. Rev. 173:5-16 (2000); Hancock, R. E., and Lehrer, R., Trends Biotechnol. 16:82-88 (1998)). In mammals, the peptides are produced and secreted by skin, mucosal surfaces and neutrophils. There are many different classes of natural host defense peptides (Zasloff, M., Curr. Opin. Immunol. 4:3-7 (1992); Zasloff, M., Trends Pharmacol. Sci. 21:236-238 (2000); Steiner, H., et al., Nature, 292:246-248 (1981); Ganz, T., et al., Eur. J. Haematol. 44:1-8 (1990); Tang, Y. Q., et al., Science 286:498-502 (1999); Ganz, T., et al., J. Clin. Invest. 76:1427-1435 (1985); Landon, C., et al., Protein Sci. 6:1878-1884 (1997); Zhao, C., et al., FEBS Lett. 346:285-288 (1994); Peggion, E., et al., Biopolymers (Peptide Science) 43:419-431 (1998); Dempsey, C. E., Biochim. Biophys. Acta 1031:143-161 (1990)), but, in general, most contain between 20-40 amino acid residues and adopt an amphiphilic secondary structure as shown in FIG. 1.
Although host defense peptides are found in a wide variety of species and are composed of many different sequences, their physiochemical properties are remarkably similar. They adopt an amphiphilic architecture with positively charged groups segregated to one side of the secondary structure and hydrophobic groups on the opposite surface. For example, magainin and some of the other naturally occurring antibacterial peptides contain positively charged amino acids and a large hydrophobic moment. Although these peptides exhibit considerable variation in their chain length, hydrophobicity and distribution of charges, they have a high propensity to adopt α-helical conformations in a hydrophobic environment, e.g., a cell surface or a natural or synthetic membrane (Oren, Z., and Shai, Y., Biopolymers (Peptide Science) 47:451-463 (1998)). The periodic distribution of hydrophobic and hydrophilic side chains in their amino acid sequences allows the segregation of the hydrophobic and hydrophilic side chains to opposite faces of the cylinder formed by the helix. These structures can be described as facially amphiphilic regardless of whether the secondary structure is a helix or sheet type fold. In fact, it is the overall physiochemical properties that are responsible for the biological activity of these peptides and not the precise sequence (Zasloff, M., Curr. Opin. Immunol. 4:3-7 (1992); Zasloff, M., Trends Pharmacol. Sci. 21:236-238 (2000); Hancock, R. E., and Lehrer, R., Trends Biotechnol. 16:82-88 (1998); DeGrado, W. F., et al, J. Amer. Chem. Soc. 103:679-681 (1981); DeGrado, W. F., Adv. Prot. Chem. 39:51-124 (1988); Tossi, A., et al., Biopolymers 55:4-30 (2000); Merrifield, E. L., et al., Int. J. Pept. Protein Res. 46:214-220 (1995); Merrifield, R. B., et al., Proc Natl Acad Sci (USA) 92:3449-3453 (1995)). Because the overall amphiphilicity, not the specific sequence, secondary structure or chirality, correlates best with the anti-microbial activity of these peptides, it appears that any suitably amphiphilic material (not necessarily an α-helix or β-sheet) would have anti-microbial properties.
The cytotoxic activity of these cationic and amphiphilic antimicrobial peptides is also specific for bacteria over mammalian cells. This specificity is most likely related to fundamental differences between the two membrane types. For example, bacteria have a large proportion of negatively charged phospholipid headgroups on their surface, while, in contrast, the outer leaflet of animal cells is composed mainly of neutral lipids (Zasloff, M., Nature 415:389-395 (2002)). The presence of cholesterol in the animal cell membrane also appears to reduce the activity of the antimicrobial peptides.
The bactericidal activity of the host defense peptides is very rapid, occurring within minutes after exposure of bacteria to lethal doses of peptide. Several mechanisms have been proposed for the process of cell killing. According to the carpet mechanism, host defense peptides aggregate parallel to the membrane surface (Gazit, E., et al., Biohemistry 34:11479-11488 (1995); Pouny, Y., et al., Biochemistry 31:12416-12423 (1992)), leading to thinning and, ultimately, rupture of the membrane. In the so-called barrel-stave mechanism, the bound peptides on the cell surface self-associate into transmembrane helical bundles that form stable aqueous pores in the membrane (Merrifield, R. B., et al., Ciba Found. Symp. 186:5-20 (1994)). According to a third possible mechanism (DeGrado, W. F., et al., Biophys. J. 37:329-338 (1982)), the peptides initially bind only to the outer leaflet of the bilayer, leading to an increase in the lateral surface pressure of the outer leaflet relative to the inner leaflet of the bilayer. This pressure imbalance results in translocation of the peptides into the interior of the bilayer with concomitant formation of transient openings in the membrane. Formation of these transient pores allows hydration of the polar sidechains of the peptide and leakage of cellular contents. Most antimicrobial peptides probably act by more than one of these mechanisms. Additionally, some classes may interact with periplasmic or intercellular targets (Zasloff, M., Trends Pharmacol. Sci. 21:236-238 (2000)).
In addition to the well-characterized antibacterial activity, several of the host defense peptides possess antifungal activity. Examples of mammalian, insect and amphibian peptides with demonstrated antifungal activities include defensins, protegrins, lactoferrin-B, cecropins, and dermaseptins (DeLucca, A. J., and Walsh, T. J., Antimicob. Agents Chemother. 43:1-11 (1999)). The mechanism of cytotoxic action appears to be similar to that for bacteria, leading to rapid lysis of the fungal membrane.
Several host defense peptides also possess antiviral activity. For example, several classes of host defense peptides also inhibit the replication of both DNA and RNA viruses. NP-1, a prototypic alpha-defensin, protects cells in culture from infection by herpes simplex virus-2. The block appears to occur very early in the infection cycle as the peptide prevents viral entry but does interfere with binding between the viral glycoproteins and the cellular heparin sulfate receptors (Sinha, S., et al., Antimicrob. Agents Chemother. 47:494-500 (2003)). Several other host defense peptides have been shown to have antiviral activity against herpes simplex virus-1 (Belaid, A., et al., J. Med. Virol. 66:229-234 (2002); Egal, M., et al., Int. J. Antimicrob. Agents 13:57-60 (1999)) as well as human cytomegalovirus virus (Andersen, J. H., et al., Antiviral Rs. 51:141-149 (2001)). NP-1 also inhibits adenoviral infection in cell culture (Bastian, A., and Schafer, H., Regul. Pept. 15:157-161 (2001)).
The human alpha-defensins have also been shown to inhibit the replication of HIV-1 isolates in vitro (Zhang, L., et al., Science 298: 995-1000 (2002)) and to be the active components of a soluble fraction that suppresses HIV-1 replication which is secreted from CD8 T lymphocytes isolated from long-term nonprogressing AIDS patients (Zhang, L., et al., Science 298: 995-1000 (2002)). The mechanism by which the defensins inhibit HIV replication is unknown but the block occurs early in the infection cycle at or near the time of viral entry. The antimicrobial peptides melittin and cecropin have also been reported to inhibit HIV-1 replication and it is suggested that they exert their activity by suppressing HIV gene expression (Wachinger, M., et al., J. Gen. Virol. 79:731-740 (1998)).
The mechanism of antiviral action of the host defense peptides appears not to be related to direct virucidal activity, where the integrity of the virion is disrupted, but rather at an early stage in the infection cycle during entry of the virus into the host cell.
The design of non-biological polymers with well-defined secondary and tertiary structures has received considerable attention in the past few years (Gellman, S. H., Acc. Chem. Res. 31:173-180 (1998); Barron, A. E., and Zuckermann, R. N., Curr. Opin. Chem. Biol. 3:681-687 (1999); Stigers, K. D., et al., Curr. Opin. Chem. Biol., 3:714-723 (1999)). Using these principles, investigators have designed synthetic antimicrobial peptides by idealizing the amphiphilic α-helical arrangement of sidechains observed in the natural host defense peptides, leading to a large number of potent and selective antimicrobial compounds (Tossi, A., et al., Biopolymers 55:4-30 (2000); DeGrado, W. F., Adv. Protein. Chem. 39:51-124 (1988); Maloy, W. L., and Kari, U. P., Biopolymers 37:105-122 (1995); Zasloff, M., Curr. Opin. Immunol. 4:3-7 (1992); Boman, H. G., et al., Eur. J. Biochem. 201:23-31 (1991); Oren, Z., and Shai, Y., Biopolymers 47:451-463 (1998)).
β-peptides have also provided another avenue to test and further elucidate the features required for the construction of bactericidal agents. β-peptides adopt L+2 helices, which have an approximate 3-residue geometric repeat. Thus, if polar and apolar sidechains are arranged with precise three-residue periodicity in the sequence of a β-peptide, they should segregate to opposite sides of the helix. Using this approach, DeGrado and co-workers (Hamuro, Y., et al., J. Amer. Chem. Soc. 121:12200-12201 (1999); Liu, D., and DeGrado, W. F., J. Amer. Chem. Soc., 123:7553-7559 (2001)) have designed synthetic β-peptide oligomers that are roughly equipotent in antimicrobial activity to many naturally occurring peptide antibiotics. The antimicrobial activities of these β-peptides and their specificities for bacterial cells over mammalian cells can be controlled by fine-tuning their hydrophobicities and chain lengths. Gellman and coworkers have also synthesized cyclically constrained β-peptides possessing potent antimicrobial activity and minimal activity against mammalian cells (Porter, E. A., et al., Nature 404:565 (2000)).
Non-peptidic antimicrobial polymers have also been developed. For example, suitably substituted polymers lacking polyamide linkages that are capable of adopting amphiphilic conformations have been designed and synthesized. Solid phase chemistry technology has been utilized to synthesize a class of meta substituted phenylacetylenes that fold into helical structures in appropriate solvents (Nelson, J. C., et al, Science 277:1793-1796 (1997); Prince, R. B., et al., Angew. Chem. Int. Ed. 39:228-231 (2000)). These molecules contain an all hydrocarbon backbone with ethylene oxide side chains such that when exposed to a polar solvent (acetonitrile), the backbone collapses to minimize its contact with this polar solvent. As a result of the meta substitution, the preferred folded conformation is helical. This helical folding is attributed to a “solvophobic” energy term; although, the importance of favorable π-π aromatic interactions in the folded state are also likely to be important. Furthermore, addition of a less polar solvent (CHCl3) results in an unfolding of the helical structure demonstrating that this folding is reversible.
In addition, Mandeville et al., U.S. Pat. No. 6,034,129, disclose anti-infective vinyl copolymers, wherein monomers with hydrophobic and hydrophilic side chains have been randomly polymerized to produce polymers with amphiphilic properties. These materials are produced by polymerization of hydrophobic and hydrophilic acrylate monomers. Alternately, the hydrophobic side chain is derived from a styrene derivative which is copolymerized with a hydrophilic acrylate monomer wherein an ionic group is linked to the carboxylic, acid.
Tew et al. (Tew, G. N., et al., Proc. Natl. Acad. Sci. (USA) 99:5110-5114 (2002)) disclose the design and synthesis of a series of biomimetic, facially amphiphilic arylamide polymers possessing antimicrobial activity. The arylamide polymers were designed using de novo computational design techniques.
WIPO Publ. No. WO 02/100295 discloses facially amphiphilic polyamide, polyester, polyurea, polycarbonate, and polyurethane polymers with anti-infective activity, and articles made from them having biocidal surfaces. WIPO Publ. No. WO 02/100295 is fully incorporated by reference herein in its entirety.
WIPO Publ. No. WO 02/072007 discloses a number of facially amphiphilic polyphenylene and heteroarylene polymers, including polyphenylalkynyl polymers, with anti-infective activity and articles made therefrom having biocidal surfaces. WIPO publication no. WO 02/072007 is fully incorporated by reference herein in its entirety.